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Supplementary Information

Chronic Stress Alters Spatial Representation of Place Cells and Bursting

Patterns in Mice

Mijeong Park1,3, Chong-Hyun Kim1,3, Seonmi Jo2, Eun Joo Kim4, Hyewhon Rhim1,3,

C. Justin Lee2,3, Jeansok J. Kim4, Jeiwon Cho1,3*

1Center for Neuroscience 2Center for Functional Connectomics, Korea Institute of

Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 136-791, Korea,

3Neuroscience Program, Korea University of Science & Technology, Daejeon 305-

701, Korea, 4Department of Psychology, University of Washington, Seattle, WA

98195-1525, USA

*Corresponding author: Jeiwon Cho, Ph.D., Center for Neuroscience, Korea Institute

of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 136-791,

Korea; E-mail: jeiwon@kist.re.kr

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Supplementary Methods & Materials

Plasma corticosterone hormone levels (CORT). Blood samples were collected on

days 1, 10 and 21 via decapitation in the morning to assess the basal corticosterone

hormone level 1. Blood samples from the stressed mice were taken 1 hr after the

initiation of the restraint stress in order to measure the peak level of the stress

hormone 2. The blood samples were centrifuged (1000g, 4°C, 10 min), then the

plasma samples were separated and stored at -20°C 3. Plasma corticosterone was

analyzed using the Corticosterone Enzyme Immunoassay (EIA) kit (Assay Designs,

Ann Arbor, MI) and hormone levels were measured using ELISA reader (Molecular

Devices, CA, USA).

Golgi staining. After 21 days of CRS, the stressed mice (N=4) and time-matched

control mice (N=4) underwent cervical dislocation/decapitation. The brain was

removed and fixed with Golgi staining solution in the manner specified by the FD

Rapid GolgiStain Kit (FD NeuroTechnologies, Ellicott City, MD). Coronal sections of

80 µm thickness were cut through the entire hippocampus region using a cryostat

(Microm, Germany) at -23°C. Well stained pyramidal neurons were selected and the

dendritic spines were counted in 50 µm successive segments of apical dendrites in

CA1 and CA3 regions using a microscope (OLYMPUS, BX50, 1000X magnification).

Hippocampal slice preparation and Current-clamp recording. After 21 days of

CRS, mice were deeply anesthetized with isoflurane, followed by decapitation.

Dorsal hippocampal slices (300 m thickness) were prepared from each side of one

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mouse brain. Hippocampal containing region was dissected out using cold

oxygenated (95%O2/5%CO2) artificial CSF(aCSF) (mM): NaCl 124, KCl 2.5,

NaHCO3 26, NaH2PO4H2O 1.25, Glucose 11, CaCl2H2O 0.5, MgCl2 5, pH 7.3. Slices

were kept on the surface of cell culture inserts in an incubation chamber to which

humidified oxygen was continuously supplied for storage, and at least one hour after

dissection, one or two slices were transferred to the recording chamber for the

recording. The recording aCSF has 2.5mM Ca and 1.3mM. For whole cell recording,

pyramidal neurons were selected in CA1 pyramidal cell body layer by infrared

differential interference contrast (IR-DIC) microscopy (Olympus BX51W1) with a 40x

objective. The intracellular solution has the following components (mM): K-gluconate

120, KCl 15, MgCl24, HEPES 10, MgATP4, Na3GTP 0.3, EGTA 0.1,

Phosphocreatine 7, pH 7.4 (about 300 mOsm). The traces were filtered at 2 kHz.

Histology. Upon the completion of recordings, the mice were overdosed with 2%

Avertin and a small current (10-30 µA, 10 sec) was passed through one of 4 wires in

the tetrode to mark the recording site. The mice were transcardially perfused with 10%

formalin and then brains were extracted and fixed further in 10% formalin at room

temperature. Coronal sections (50 μm) were cut through the entire hippocampus

region using a cryostat (Microm, Germany) at -23°C. Brain slices were mounted on

the slides and then stained with Cresyl Violet (Fig. 3a).

Western Blotting. After 21 days of CRS, mice were anesthetized by i.p. injection of

tribromoethanol (Avertin, 20 mg/mL). The brain was quickly excised from the skull

and submerged in ice-cold PBS. After cooling, CA1 region of the hippocampus from

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each mouse were separated and stored at -70oC until analysis. Each sample was

homogenized and lysed with RIPA buffer 20g of proteins (as estimated using the

BCA reagent; Pierce) were separated by SDS-PAGE using 10% polyacrylamide gels

and blotted onto PVDF membranes. The blots were incubated overnight at 4oC with

either rabbit anti-CAMK2 (phospho T286) antibody (1:1000, Abcam), mouse anti-

CAMK2 antibody (1:1000, Abcam). For protein loading control, each blot was

incubated with mouse anti-GAPDH antibody (1:1000, Abcam). Blots were then

washed and incubated with horseradish peroxidase-conjugated goat anti-mouse,

followed by washing and detection of immunoreactivity with enhanced

chemiluminescence (Amersham Biosciences). The band intensity was acquired and

analyzed by Image Quant LAS4000 (General Electric Company).

Reference

1. Seasholtz, A.F. & Rozeboom, A.M. Mineralocorticoid receptor overexpression

in forebrain decreases anxiety-like behavior and alters the stress response in

mice. Proceedings of the National Academy of Sciences of the United States

of America 104, 4688-4693 (2007).

2. Magarinos, A.M. & McEwen, B.S. Stress-induced atrophy of apical dendrites

of hippocampal CA3c neurons: involvement of glucocorticoid secretion and

excitatory amino acid receptors. Neuroscience 69, 89-98 (1995).

3. Pavlides, C., Nivon, L.G. & McEwen, B.S. Effects of chronic stress on

hippocampal long-term potentiation. Hippocampus 12, 245-257 (2002).

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Figure S1. Experimental designs. (a) Photograph of the restraint stress. Diagram

of the experimental designs for (b) corticosterone hormone measurement, Golgi

staining, western blot, In vitro electrophysiology, (c) hidden platform Morris water

maze (MWM) test, (d) visible platform MWM, and (e) the place cell recording

experiment in freely moving mice.

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Figure S2. General effects of CRS. (a) Body weight across 21 days. (b) CORT

levels from three sampling time points. (c) Photomicrograph of transverse

hippocampal section with Golgi staining (100X); Apical dendritic branches of CA1

and CA3 pyramidal neurons (1000X). Scale bar = 50 μm. (d) Dendritic spine

numbers from CA1 (N=14 cells from 4 control mice and N=13 cells from 4 stressed

mice) and CA3 (N=18 cells from 4 control mice and N=14 cells from 4 stressed mice)

pyramidal neurons. All values are presented as the mean+SEM (One-way repeated

ANOVA, Unpaired two-tailed t-test, Mann-Whitney U test, *P < 0.05, **P < 0.01).

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Figure S3. Action potential firing properties induced by current injection of

hippocampal CA1 pyramidal neuron. (a) Resting membrane potential (mV) of

dorsal CA1 neurons (P = 0.46). (b) Whole-cell membrane capacitance (pF) of dorsal

CA1 neurons (P = 0.46). (c) Number of action potential firings induced by 250 msec

depolarizing current injection into dorsal CA1 neurons, starting from 0 to 800 pA by

200 pA steps. Inlet traces are the example traces overlapped made at five different

current injections. All samples were collected from dorsal CA1 neurons (n=14) in

control mice and dorsal CA1 neurons (n=17) in stressed mice (P’s > 0.9). (d) Input-

Output relationships of basal evoked synaptic transmission. Left, field EPSP slopes

in response to six-step incremental stimulation current strengths. Right, linear

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regression value of slopes from each slice of the left I-O plot (P = 0.11). (e) Paired-

pulse ratio of fEPSP responses. The ratio of the 2nd fEPSP slope over the 1st fEPSP

slope was measured at CA3 to CA1 synapses in hippocampal slices (P’s > 0.1). All

values are presented as the mean+SEM (Unpaired two-tailed t-test).

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Figure S4. Western blot analysis of the effect of CRS on the αCaMK2 and

phospo-αCaMK2 in CA1 hippocampal region. Representative immunoblotting

bands and pixel volume ratio of (a) αCaMK2/GAPDH and (b) phospo-

αCaMK2/GAPDH of CA1 hippocampal region from control (n=5) and stressed (n=6)

mice. All values are presented as the mean+SEM (Unpaired two-tailed t-test, *P <

0.05).

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Figure S5. Hidden platform and visible platform Morris water maze tasks. (a)

Swimming speed during acquisition and reversal learning periods. Two groups

showed significant difference in the swim speed during acquisition period (day1, t(132)

= 6.45, P < 0.01; day2, t(132) = 6.29, P < 0.0; day4, t(132) = 5.37, P < 0.01). (b,c)

Reversal probe tests (b) The percent time spent swimming in 4 quadrants. The

stressed mice spent more time searching for the opposite quadrant (acquisition

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target quadrant) than control mice (F(1,31) = 0.16, P = 0.05, main effect of group; F(1,93)

= 2.1, P = 0.098, main effect of group x day interaction; t(31) = -2.18, P = 0.046, in the

opposite quadrant). T, target quadrant (reversal target quadrant); R, right quadrant; L,

left quadrant; O, opposite quadrant (acquisition target quadrant). (c) The platform

crossing number (t(31) = 0.64, P = 0.52). (d) Swimming speed during the retention

test (trial 9) in the visible platform test (see Fig 2d). All values are presented as the

mean+SEM (One-way repeated ANOVA, Unpaired two-tailed t-test, *P < 0.05, **P <

0.01).

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Figure S6. Examples of cue dependency between session 1 and session2

during 3 recording sessions. In session 2 (local cue rotation), unit # 1 to 5

(Rotation) rotated their place fields to follow the local cue rotation and unit # 6 to 10

(Stay) maintained their place fields in the same position as session 2. The place

fields of unit # 11 to 13 (Remapping) showed remapping between session 1 and 2.

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When place fields were compared between session 1 and 3, most units showed

similar place fields. The number on top right of each place map represents peak FR.